The present invention relates to a device and method for determining the presence of antigens in biological fluids.
Immunologic diagnostic tests are widely used for the detection of body fluid antigens, hormones, infectious agents, and serum antibodies. Immunoassays generally fall into the following two categories. First, antibody-antigen precipitation tests, such as radial immunodiffusion, hemagglutination, and coated latex particle agglutination. Second, labeled-reagent tests, such as radioimmunoassay, and enzyme-linked immunoassay.
The precipitation type tests have the advantage of being performed manually and are commercially used in disposable kits which are read visually and do not require an instrument. The reading from a precipitation type immunoassay is usually expressed as the presence or absence of an agglutination reaction at each of a series of known dilutions of the test sample or competing antigen. The disadvantages of the precipitation type tests is that they are much less sensitive than the labeled reagent assays, require time consuming incubation steps, and are susceptible to subjective error in visual identification of a precipitation reaction.
Labeled reagent immunoassays are quantitative and highly sensitive but, nevertheless, have certain disadvantages. Radioimmunoassays employ radioactive tracers and, therefore, require a gamma radiation detection instrument. The radioactive tracers have a short shelf life, pose a health hazard to the technician and have been subject to restrictive legislation. Enzyme-linked immunoabsorbant assays (ELISA) use reagents labeled with an enzyme. The enzyme is detected by its reaction with a substrate to yield a product that can be easily measured (for example by formation of a color). The ELISA does not require radioactive materials and uses reagents with a long shelf life.
The ELISA assay beings with the binding of a reference reagent to a solid phase support, such as the bottom of a plastic well. Test fluid, mixed with enzyme-labeled reagent, is reacted with the bound reference reagent. Through a number of dilution, incubation and washing steps (as many as fourteen), bound and free reagents are separated, and a color forming reaction is initiated. The intensity of the color formed at different serial dilutions provides the quantitative measure. The standard ELISA takes between four and twelve hours to perform. Consequently, the major disadvantage of the ELISA is the large number of dilution, incubation, and washing steps, which are time consuming and subject to error.
The device of the present invention is used to overcome many of the difficulties associated with the above-discussed type of assay. Specifically, the use thereof results in an improved ELISA which requires no dilution, no washing steps, and one short incubation period. The assay is in the form of a dry, layered test strip which forms a color reaction when exposed directly to the test fluid. The strip automatically performs the dilution steps required for quantitation and separates the bound antibody from the free antibody. The color reaction can be read visually, or with an instrument, such as a spectrophotometer. In addition, the device of the present invention can be fabricated in strip form and employed as a dipstick for rapidly detecting an antigen, such as a drug or hormone in urine. An example of a specific application would be the rapid detection of a drug overdose in the Emergency Room, or as a home pregnancy test.